The present invention relates to an optical method for high-sensitivity detection of fluorescent molecules based on the use of a highly focused light beam and light-induced fluorescence spectroscopy, to an apparatus for high-sensitivity detection of fluorescent molecules comprising a light source and a fluorescence detector, to a method for the production of a flow cell for high-sensitivity detection of fluorescent molecules, as well as to use of said method, apparatus or flow cell in combination with a microscope.
Techniques based on miniaturised chemical separation have made possible the analysis of the contents of individual cells (O. Orwar, H. A. Fishman, N. Ziv, R. H. Scheller, R. N. Zare, Anal. Chem., 67, 4261 (1995)), and individual subcellular organelles (D. T. Chiu, S. J. Lillard, R. H. Scheller, R. N. Zare, S. E. Rodriguez-Cruz, E. R. Williams, O. Orwar, M. Sandberg, J. A. Lundqvist, Science in press). However, there is a need to analyse the contents of ever smaller sample volumes and even monomeric units cleaved off from single biopolymers such as RNAs, DNAs, and proteins. In order to render this possible, it is necessary to develop techniques with sensitivities approaching the inverse of Avogadro""s constant, NA (6.0221xc3x971023 molxe2x88x921)
The use of microcolumn separation techniques, such as capillary electrophoresis, capillary electrochromatography, and microcolumn high performance liquid chromatography, for the compositional analysis of various types of samples, especially in the area of biomedical research and in the pharmaceutical industry, has experienced tremendous growth during the last twenty years. Since these techniques are particularly useful for the analysis of ultra-small sample volumes, 10xe2x88x926 to 10xe2x88x9221 litres, that often contain trace amounts of analytes, severe demands are placed on detection sensitivity.
The possibility to detect very small quantities of biologically important molecules is of great interest in many fields, such as molecular biology, medical diagnosis, drug development and forensic analysis. Of particular interest is often the detection of antibodies, antigens, hormones, enzymes, proteins, peptides, amino acids or nucleic acids present in a sample. However, these samples often contain very small amounts of the molecules in question and they are therefore difficult to detect adequately. It is often necessary to amplify the material to obtain greater quantities before detection. In the case of e,g. DNA, this amplification is most frequently made by means of polymerase chain reaction (PCR), which duplicates DNA sequence of interest However, amplification of the molecules to be detected is not always desirable since it may, for example, lead to the introduction of substances contaminating the sample. Hence, there is a demand for techniques enabling direct detection of small amounts of a given substance. There are already some techniques available, and most of these are based on optical detection methods and on the use of different spectroscopy methods.
In 1961 came the first report on single-molecule studies in solution (B. Rotman, Proc. Natl. Acad. Sci., USA, 47,1981 (1961)). This study also has biological significance since the presence of a single enzyme molecule could be detected using a fluorogenic substrate. In 1976 a single antibody tagged with 80-100 fluorescein molecules could be detected using evanescent-wave excitation (T. Hirschfeld, Appl. Opt. 15, 2965 (1976)). Since then, much has been done in this field. One of the most promising techniques for sensitive detection is laser-induced fluorescence, mainly applied in two different set-ups: detection within a focused laser beam and detection in a near-field scanning optical microscope. Other techniques, such as nuclear magnetic resonance, electrochemistry, cavity ring-down spectroscopy have also been proposed for single molecule studies. Also, the use of biosensors in chemical separations have made it possible to distinguish single biomolecules (O. Orwar, K. Jardemark, I. Jacobson, A. Moscho, H. A. Fishman, R. H. Scheller, R. N. Zare, Science, 272, 1779 (1998)).
Methods based on laser-induced fluorescence have been demonstrated to have the ability to detect a single fluorescent molecule in solution. However, the known methods are diffusion-limited and can be employed only for samples containing a large amount of fluorescent molecules. Therefore, the sampling efficiency, i.e. the number of fluorescent molecules detected over the total amount of fluorescent molecules present in the solution, is extremely small, on the order of 10xe2x88x926 or even less. In one commonly employed embodiment of single-molecule detection in solution, a drop containing the fluorescent molecules is placed on a coverslip (S. Nie, D. T. Chiu, R. N. Zare, Anal. Chem., 67, 2849 (1995) and R. Riegler, U. Mets, J. Widengren, P. Kask, Eur. Biophys. J., 22, 169 (1993) and S. Nie, D. T. Chiu, R. N. Zare, Science, 266, 1018 (1994)). Single-molecule fluorescence is then collected and detected in a confocal fluorescence microscope set-up. With this technique it is, however, difficult to accomplish detection of molecules separated by a microchemical fractionation technique.
Detection of single molecules has also been achieved in capillary structures, both coupled to separation devices and as stand-alone flow cells (Y-H Lee, R. G. Maus, B. W. Smith, J. D. Winefordner, Anal. Chem., 64, 4142 (1994)). Also in these cases, however, detection has been performed in solutions containing a large excess of the fluorescent molecule over the actual detected number of molecules. Typically, 10xe2x88x929 to 10xe2x88x9212 M of fluorescent solutes are introduced into the system in solution volumes of from 10xe2x88x926 to 10xe2x88x923 1. Thus, again sampling efficiencies on the order of 10xe2x88x926 to 10xe2x88x9212 are obtained.
In the last decade, there has been rapid development in high-resolution optical and electro-optical techniques, driven by the need to understand biochemical and biophysical processes in greater detail. For example, confocal microscopy and two-photon microscopy have provided striking images on the workings of cellular machinery, such as the dynamics of intracellular calcium ion and the localisation of single serotonin-containing granulae in RBL cells (see egg. B J. Bacskai, P. Wallen, V. Lev-Ram, S. Grillner, R. Y. Tsien, Neuron, 14, 19-28 (1995) and S. Maiti, J. B. Shear, R. M. Williams, W. R Zipfel, W. W. Webb, Science, 275, 530-532 (1997)). Higher optical resolutionsxe2x80x94as high as 12 nmxe2x80x94are obtained in near-field spectroscopic probes, wherein it is possible to reach, or even bypass the Abbe diffraction limit (E. Betzig, J. K. Trautman, T. D Harris, J. S. weiner, R. L Kostelak, Science, 251, 1468 (1991)). The manipulation of single organelles and even single biomolecules has been made possible by optical trapping, and this technique has been applied to a wide range of interesting biological problems (A. Ashkin, Phys. Rev. Lett., 24 (4), 156 (1970) and K. Svoboda, S. M. Block, J. Annu. Rev. Biophys. Biomol. Struct., 23, 247-285 (1994) and D. T. Chiu, A. Hsiao, A. Gaggar. R. A. Garza-Lopez, O. Orwar, R. N. Zare, Anal. Chem., 69, 1801-1807 (1997)).
As stated above, techniques that can detect a single molecule rapidly moving in solution are based almost exclusively on optical methods. By using lasers which produce spatially and temporally coherent bundles of monochromatic light, a tightly focused diffraction-limited laser spot can be obtained with appropriate optics.
If detection is made through a pinhole or a narrow slit, in a confocal detection arrangement, an extremely small laser probe volume can be created on the order of about 5xc3x9710xe2x88x9216 1. In this way, an extremely narrow depth-of-focus is obtained. The confocal advantage includes extremely low background scattering from Rayleigh and Raman events, where the intensity has an inverse quadruplicate dependence on laser wavelength, a linear dependence on laser power, and is unsaturable. Molecular fluorescence on the other hand, is saturable and its dependence on laser irradiance is exponential. Using confocal fluorescence microscopy, it has been demonstrated that single highly fluorescent molecules such as laser dyes can be detected with high signal-to-noise ratios (S. Nie, D. T. Chiu, R. N. Zare, Anal. Chem., 67, 2849 (1995) and R. Riegler, U. Mets, J. Widengren, P. Kask, Eur. Biophys. J., 22, 169 (1993) and S. Nie, D. T. Chiu, R. N. Zare, Science, 266, 1018 (1994)). However, also according to this technique, high concentrations of the molecules to be detected are necessary. The sample containing the molecules to be detected is placed in a chamber or on a coverslip and detection of a single molecule is therefore diffusion limited. In this random and chaotic detection format, there exists no externally applied force to place the molecules in the laser probe volume. This means that the sampling efficiency, defined as the number of detected molecules over the total number of molecules present in the sample, typically is on the order of 10xe2x88x926 or less. To fulfil the criteria of volume-independent detection limits approaching NAxe2x88x921, this ratio needs to be close to unity. If this criteria is fulfilled it is possible to detect and probe a molecule even if the sample solution only contains a single molecule.
Reports on experiments using laser-induced fluorescence detection in capillaries have demonstrated exquisite sensitivities (Y-H Lee, R. G. Maus, B. W. Smith, J. D. Winefordner, Anal. Chem., 64, 4142 (1994) However, the instrumentation and analysis (deconvolution algorithms) have seen difficult to implement in some cases and most of the techniques does not have the desired concentration detection limits since the probe volume is much smaller than the dimensions of the capillaries Although a single molecule can be detected once it resides in the probe volume, most molecules do not traverse the probe volume and are thus missed.
The object of the present invention is to provide a simple method and apparatus that enables high-sensitivity detection of fluorescent molecules, and in particular ultra-sensitivity detection of single fluorescent molecules in a flowing stream, said detection having a sampling efficiency close to unity. This means that it will be possible to detect a single molecule present in a solution regardless of the volume of the solution.
Thus, the present invention relates to an optical method for high-sensitivity detection of fluorescent molecules based on the use of a highly focused light beam and light-induced fluorescence spectroscopy characterised in that
(I) a sample comprising at least one fluorescent molecule is made to flow through at least one flow cell consisting of at least one channel structure comprising at least one constricted region, said at least one constricted region having a cross-section of a dimension corresponding to the size of a tightly focused light spot close to or at the diffraction limit and extremely thin, transparent walls,
(II) at least one light beam is focused close to or at the diffraction limit inside said at least one constricted region and thus exciting any fluorescent molecules present in the sample volume passing through said at least one constricted region, and
(III) the fluorescence emitted when a fluorescent molecule or a group of molecules passes through said at least one constricted region and is excited is detected.
The invention also relates to an apparatus for high-sensitivity detection of fluorescent molecules comprising at least one light source and at least one fluorescence detector, characterised in that it further comprises at least one flow cell consisting of at least one channel structure comprising at least one constricted region, said at least one constricted region having a cross-section of a dimension corresponding to the size of a tightly focused light spot close to or at the diffraction limit and extremely thin, transparent walls, said at least one channel structure being adapted to accommodate the sample comprising the molecule or molecules to be detected.
Furthermore, the invention relates to a method for the production of a flow cell for use in high-sensitivity detection of fluorescent molecules characterised in that a channel structure is obtained at an appropriate method, and at least one region of said channel structure is then heated until the melting point of the material constituting the channel structure is reached, and in that the channel structure finally is pulled in order to lengthen the melted region and thus make it thinner until it has a dimension corresponding to the size of a tightly focused laser spot close to or at the diffraction limit, said material constituting the channel structure being transparent or turning transparent during the heat treatment.
Finally, the invention relates to use of the above mentioned method and/or apparatus for high-sensitivity detection of fluorescent molecules, and/or a flow cell produced according to the above mentioned method in combination with a microscope, preferably a confocal fluorescence microscope, and most preferably a scanning confocal fluorescence microscope.
The characterising features of the invention will be evident from the following description and the appended claims.
According to the present invention, it is thus possible to detect single fluorescent molecules, as well as groups of fluorescent molecules. However, it is also possible to detect non-fluorescent molecules by tagging them with a fluorescent or a fluorogenic compound before detection,
Beside detection of fluorescent molecules, it is also possible according to the invention to detect small fluorescent particles.
The light beam used according to the invention preferably has a wavelength of approximately 200-1500 nm. The light source is preferably a laser, and most preferably an argon ion laser.
It is possible to use more than one light source, and it is then advantageously if each light source emits light at a different wavelength. This enables simultaneous detection of molecules with different fluorescence spectral properties.
The light beam is focused to a spot close to or at the diffraction limit in the constricted region of the flow cell through use of appropriate means. Said means may e.g. be a high-numerical aperture microscope objective (100xc3x97). 
The excitation of the fluorescent molecules may be achieved either by a single-photon process or by a multi-photon process. Preferably, the excitation is made in a two-photon or multi-photon mode.
The channel structure used according to the present invention is preferably a capillary, and most preferably a fused silica glass capillary. It may also be a channel etched into a chip. Furthermore, it is advantageously to use several channel structures parallelly. The channel structures are then preferably arranged in a co-planar mode. This feature is advantageous especially for high throughput screening applications.
The flow channel may further form an integrated and continuous part of a glow injection analysis system or a separation system, such as a system for capillary electrophoresis, capillary electrochromatography, liquid chromatography or gas chromatography. For these purposes the flow channel may be packed with a suitable material, such as beads.
The dimensions of the constricted region of the flow cell according to the invention is made to match the size of the volume being illuminated by the light. The constricted region preferably has an inner diameter (id.) of approximately 0.2-8xcexcm, and an outer diameter (o.d.) of approximately 0.4-40xcexcm. Since the constricted region of the flow cell is physically narrower than the rest of the flow cell, the solution cravelling through the flow cell is focused in the constricted region. Since only a small portion of the flow cell is constricted, the flow cell can accommodate large sample volumes. This possibility to handle large sample volumes is an important and distinctive feature of the present invention. The concentration sensitivity is several orders of magnitude higher than previous accounts of single molecule detection. It is possible to detect a single molecule almost independent of the sample volume in e.g. a flow injection analysis scheme. Since many biological samples are concentration limited rather than volume limited, this aspect of the invention is important. Once the sample is introduced in the flow cell, the probability of detecting the molecules is almost unity since the dimensions of the probe volume and the constricted region are well matched, and all molecules will traverse the probe volume with knowledge of the total sample volume injected into the flow cell, this can yield sample concentration without calibration. This is also an important and distinctive feature of the present invention because it abolishes the need to detect analytes in standard solutions of known concentrations. Hence, quantitative analyses can be performed at lower cost and higher sample turnover rate than conventional technologies.
In order to maintain the quality of the light beam and to minimise spherical and other aberrations, the channel walls are made extremely thin, on the order of a few microns or less. These thin walls minimise the cylindrical lensing effects observed for capillaries with walls of regular thicknesses.
It is advantageously to place the constricted region in a medium with a refractive index close to that of the material constituting said constricted region This medium is preferably oil or water, or water supplemented with appropriate additives This results in a higher optical tuning of the system, by avoiding the light passing through a medium with a refractive index of 1.
Furthermore, it is advantageously that the channel structure comprises more than one constricted region. It is then possible to measure the emitted fluorescence at different constricted regions and cross-correlate the data in order to improve the probability of identifying a true detection event from a chaotic background event.
The detection of the emitted fluorescence is preferably made by means of a highly sensitive photon detector, such as a single photon counting diode or a photon counting photomultiplier tube, or a highly sensitive photon counting charge coupled device., a VIM camera or a streak camera. It is possible to perform the detection either at a single wavelength or in a multicolour format. It is further possible to perform the detection in either a confocal or a non-confocal mode. The confocal mode is preferred for single-photon excitation and the non-confocal mode is preferred for multi-photon excitation.
The flow cell used according to the invention forms preferably an integrated and continuous part of a flow injection analysis system or a separation system, such as a capillary electrophoresis, capillary electrochromatography, liquid chromatography, or gas chromatography system.
A particularly interesting field for application of the present invention is analysis of single DNA, RNA, and protein molecules by sequential cleavage and detection of the fluorescently labelled monomeric units. In principle, the methodology can be applied for such analyses where the biomolecules of interest have been extracted from a single cell or even a single organelle.
Because most biologically relevant molecules do not contain any features for their sensitive detection in fluorescence, they need to be modified chemically to become fluorescent Highly selective reagents that renders biomolecules fluorescent are available (see e.g. Handbook of Molecular Probes and Research Chemicals sixth edition, 1996, by Richard P. Haugland). These fluorescent or fluorogenic reagents can be of a high specificity and react only with a single molecule, for example, fluorescently labelled antibodies or they can be general in nature and react with specific functional groups present in many different types of molecules. Examples of the latter include aldehydic reagents (e.g. o-ophthalaldehyde, and 2,3-naphthalenedicarboxaldehyde) for amino acids, and peptides that in the presence of nucleophilic co-reagents such as cyanide ion or xcex2-mercaptoethanol form highly fluorescent isoindolyl derivatives, and bimanes (e.g. monobromobimane, and monochlorobimane) that form highly fluorescent derivatives upon conjugation with thiol-containing molecules.
There are many different ways in which analytes of interest can be converted into fluorescent derivatives. It is, for example, possible to react them with a fluorescent or fluorogenic reagent prior to introduction of the sample into the capillary flow cell. It is also possible to react the analytes with a fluorogenic compound on-column, i.e. within the flow cell. This can be achieved simply by filling the capillary with the fluorogenic reagent of choice, and because the reagent does not fluoresce in itself, this procedure does ideally, not cause any interference. This procedure of analyte derivatisation has been successfully adopted to the analysis of single atrial gland vesicles isolated from the mollusc Aplysia Californica (D. T. Chiu, S. J. Lillard, R. H. Scheller, R. N. Zare, S. E. Rodriguez-Cruz, E. R. Williams, O. Orwar, M. Sandberg, J. A. Lundqvist, Science in press). In the case of single-cell analysis, the fluorescent or fluorogenic reagent can be either microinjected or electroporated directly into a biological cell before it is lysed and introduced into the capillary. It is also possible to modify the flow cell in such a way that it can accommodate a biological cell in a compartment or reaction chamber that has been injected into said flow cell. Such a biological cell reaction chamber can, for example, be formed by two constrictions in the capillary where the distance between the constrictions is matched to accommodate a single biological cell. It is also feasible to connect capillaries or electrodes to the cell reactor for chemical manipulation and electroporation of the content of the biological cell.
Because of the following unique properties of the present invention;
1. Single-molecule detection capabilities
2. Calibration-free analysis
3. Extremely high concentration sensitivity
4. Compatible to separation techniques
5. Enclosed system
it can be applied to a wide range of diagnostic and analytical applications, for which current technologies are impossible or extremely difficult to implement. Examples of such applications given below are just some examples, and does not limit the applicability of the present invention. These applications include measurement of single bacterial or viral particles in body fluids, or dietary products, and determination of the contents of single cells. In the forensic sciences it can be used for identifying DNA extracted from single cells In situations where e.g. blood has been mixed or pooled from several individuals, this represents a tremendous advantage over the use of the polymerase chain reaction (PCR), which is difficult to implement in such situations. In the area of bedside patient surveillance it might be used to measure inflammatory response proteins, indicators of intravasal chock, prechock indicators, and coagulation proteins etceteras.
The technology can also be used for determination of indicators of myocardial infarction, cardiac enzymes, etceteras. In clinical diagnostics, it can also be used for determination of DNA, RNA, bacteria, viruses, and immunoglobulins etceteras. It can further be used for detection and quantitation of immunoglobulin titers in patient serum, HIV, hepatitis, borrelia, and autoimmune markers. The present invention can also be used in the area of process analytical chemistry for quality control and product assurance, in particular of pharmaceutical formulations. It might also be well-suited for drug screening purposes including high throughput screening in a multiplexed format.